U.S. pat. appl. 2002/0097489 discloses a microscope involving the evanescent illumination of a specimen. The microscope comprises a white-light source whose light is coupled into the specimen slide via a slit diaphragm through the microscope objective for purposes of evanescent illumination. The illumination light propagates itself in the specimen slide as a result of total internal reflection, a process in which the specimen is only illuminated in the area of the evanescent field that projects from the specimen slide. Microscopes of this type are known under the designation TIRFM (Total Internal Reflection Fluorescent Microscope).
The z-resolution of TIRFMs is outstanding due to the fact that the evanescent field projects only about 100 nm into the specimen.
German pat. appl. DE 101 08 796 A1 discloses a high-aperture objective, especially for TIRF applications. The objective consists of a first lens having positive refractive power, a second lens having negative refractive power, whereby the focal length ratio between the two lenses lies within the range from −0.4 to −0.1 and the total refractive power is greater than zero. Moreover, the objective comprises two positive lenses whose diameter-to-focal length ratio is greater than 0.3 and smaller than 0.6. Furthermore, the objective comprises a negative lens and a collective lens, whereby the negative lens faces the front group and the focal length ratio of the negative lens to the collective lens lies between −0.5 and −2.
German pat. appl. DE 102 17 098 A1 discloses an incident-illumination arrangement for TIRF microscopy. This incident-illumination arrangement comprises a source of illumination that, during operation, emits a polarized illuminating bundle of rays that propagates itself at an angle relative to the optical axis, and a deflection device that deflects the illuminating bundle of rays and couples it into the objective parallel to the optical axis. With this incident-illumination arrangement, it is provided that the illuminating bundle of rays emitted by the source of illumination has s-polarization and p-polarization directions having a phase differential and the deflection device reflects the illuminating bundle of rays x times, wherein x=(n×180°−d)/60°.
German pat. appl. DE 101 43 481 A1 discloses a microscope for TIRM (Total Internal Reflection Microscopy). The microscope has a housing and an objective. The illumination light emitted by an illumination device can be coupled in via an adapter that can be slid into the microscope housing.
U.S. pat. appl. 2004/0001253 discloses a microscope with an optical illumination system that allows a simple switchover between evanescent illumination and reflection illumination. The illumination system comprises a laser light source whose light is coupled into an optical fiber. An outcoupling optical system is also provided that focuses the light emerging from the fiber into a rear focal point of the microscope objective. The optical fiber can be moved in a plane perpendicular to the optical axis of the microscope objective.
German pat. appl. DE 102 29 935 A1 discloses a device for coupling light into a microscope. There, laser light is directed at the preparation in the plane of the illuminated field diaphragm through a light-conductive fiber coupler configured as a slide. The invention is particularly well-suited for the TIRF method.
In scanning microscopy, a specimen is illuminated with a light beam so that the detection light emitted by the specimen can be observed as reflection or fluorescent light. The focus of an illuminating bundle of rays is moved in a plane of the specimen by means of a controllable beam deflector, generally by tilting two mirrors, whereby the deflection axes are usually positioned perpendicular to each other, so that one mirror deflects in the x direction while the other deflects in the y-direction. The mirrors are tilted, for example, employing galvanometric actuating elements. The power of the detection light coming from the object is measured as a function of the position of the scanning beam. Normally, the actuating elements are equipped with sensors to ascertain the actual position of the mirror. Especially in confocal scanning microscopy, an object is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light from the source is focused onto a pinhole diaphragm—the so-called excitation diaphragm—a beam splitter, a beam deflector to control the beam, a microscope optical system, a detection diaphragm and the detectors for picking up the detection or fluorescent light. The illumination light is coupled in by a beam splitter. Via the beam deflector, the fluorescent or reflection light coming from the object returns to the beam splitter, passes through it and is subsequently focused onto the detection diaphragm behind which the detectors are located. This detector arrangement is called a descan arrangement. Detection light that does not stem directly from the focus region takes a different light path and does not pass the detection diaphragm, so that point information is obtained that yields a three-dimensional image as a result of the sequential scanning of the object with the focus of the illuminating bundle of rays. For the most part, a three-dimensional image is attained by means of layer-by-layer image data acquisition.